The diagnosis of infectious disease has traditionally relied upon microbiological culture methods to identify the causative organism and determine the appropriate antimicrobial treatment. This has remained so despite recent advances in molecular and immunological diagnostics. While the development of rapid and automated methods has served to increase the efficiency of microbiological analysis, traditional quantitative culture methods remain critical for definitive diagnosis of urinary tract and other infections (Baron & Finegold, Diagnostic Microbiology, 8th ed., C. V. Mosby, [1990], p. 253).
After proper specimen collection and transport, the laboratory professional must determine which of a multitude of culture media are most appropriate to use with the culture at hand. It is important to consider the type of specimen (e.g., urine, blood, sputum, etc.), and the most commonly isolated organisms associated with disease or infection at the site of specimen collection. The time and cost necessary to achieve a final diagnosis also must be borne in mind.
With respect to the type of specimen, there are considerations related to the normal flora from which the pathogens must be differentiated. This is particularly true for fecal, rectal, vaginal, buccal and other samples which commonly contain a characteristic background flora. Urine, a fluid which is normally sterile when excreted from the kidneys, often becomes contaminated with flora from the urethra, urethral opening and skin. Indeed, as voided, urine is by no means sterile. The first voided 10 ml volume of urine can contain up to 10.sup.4 organisms per ml, due to the dislodgement of bacteria from the urethra This necessitates the differentiation of normal flora contaminants from the infecting organism(s).
As with most body sites, the normal urethra supports a characteristic normal flora. In females, the organisms comprising the normal flora vary with age and health. In premenarchal females, 66% of the organisms are aerobic coryneforms, lactobacilli, and coagulase-negative staphylococci. Streptococci are often present also. In women of reproductive age, lactobacilli are the most common isolates (Clarridge et al., "Laboratory Diagnosis of Urinary Tract Infections," Cumitech 2A, p. 1, American Society for Microbiology, 1987). In post-menopausal women, there is a marked increase in the number of anaerobes, particularly Bacteroides melaninogenicus (Clarridge et al., p. 1 ). Other organisms, such as mycoplasmas and low densities of enteric gram-negative rods may also be recovered from the urethra of healthy women (Clarridge et al.).
In males, less indigenous flora is isolated from urine. Coagulase-negative staphylococci, enterococci (i.e., group D streptococci), coryneforms, and mycoplasmas may be isolated from the urethra and urine of healthy men (Clarridge et al., supra). Table 1, lists the commensal flora (i.e., normal flora) associated with the human urinary tract.
TABLE 1 ______________________________________ Commensal Flora Associated With The Urinary Tract* Resident Flora of the Urethra ______________________________________ Coagulase-negative Staphylococci Viridans and Non-Hemolytic Streptococci Lactobacilli Corynebacterium sp. (diphtheroids) Neisseria (non-pathogenic species) Transient Gram-Negative Aerobes (including Enterobacteriaceae) Anaerobic Cocci Propionibacterium sp. Anaerobic Gram-Negative Cocci and Bacilli Commensal Mycobacterium sp. Commensal Mycoplasma sp. Occasional Yeasts ______________________________________ *Koneman et al., Color Atlas and Textbook of Diagnostic Microbiology, 4th edition, (p. 79) (J. B. Lippincott Co., 1992); Baron & Finegold, Diagnostic Microbiology, 8th ed., pp. 253-262 (C. V. Mosby, 1990); and Power & McCuen, Manual of BBL .RTM. Products and Laboratory Procedures, 6th ed., pp. 48-49 (Becton Dickinson Microbiology Systems, 1988).
Because of the associated normal flora and the desire to identify pathogenic organisms, methods of urine collection have been developed which minimize the chances of contamination, including the clean-catch midstream sample, careful catheterization, suprapubic aspiration, bladder washout, and cystoscopy. In situations where the patient cannot or will not provide a clean-catch sample, suprapubic aspiration is the method of choice (e.g., infants).
Urinary tract infections (UTI's) are among the most common infections in humans. It has been estimated that approximately 20% of all women will experience at least one UTI, with the incidence increasing with age (Baron & Finegold, Diagnostic Microbiology, 8th ed., p. 254, (C. V. Mosby, 1990)). UTI diagnosis is among the most frequent clinical investigation, with infections of the urinary tract second in frequency only to upper respiratory infections. Indeed, the requests for bacteriuria detection far exceed those for respiratory pathogen detection (Pezzlo, "Detection of Urinary Tract Infections by Rapid Methods," Clin. Microbiol. Rev., 1:268 (1988)). Overall this represents a major cost to laboratories.
The risk of UTI's is significantly increased for patients with indwelling catheters, to the point where it is highly predictable that they will eventually develop at least one UTI. With the ever-increasing number of patients in hospitals and nursing homes with long-term indwelling urinary catheters, this represents a large patient population. Even short-term catheterization presents a significant risk as there is a 20% chance that hospitalized patients with short-term catheters will develop UTI's (Baron and Finegold, supra). Indeed, the National Nosocomial Infections Study (NNIS) conducted by the Centers for Disease Control (CDC) reported that 5% to 6% of all hospitalized patients acquire nosocomial infections. It is estimated that this extends the patient's hospital stay by about 3.2 days and adds approximately $1800 to the direct costs (in 1986 figures). This amount does not take into consideration such factors as physician charges, loss of productivity, and costs associated with deaths (at least 1% of nosocomially infected patients die as a direct result of their nosocomial infection and contribute to the deaths of an additional 2-3% of infected patients).
Most UTI's are acquired by contamination of the urinary tract with the patient's fecal matter. Thus, the members of the Enterobacteriaceae and other organisms present in the patient's gastrointestinal tract are responsible for the majority of UTI's, with E. coli causing the greatest number of infections. The establishment of the gastrointestinal tract as the usual reservoir for UTI's is supported by the observation that the distribution of E. coli serotypes in UTI's corresponds closely with their relative abundance in the affected patient's gut (C. M. Kunin, Detection, Prevention and Management of Urinary Tract Infections, Lea & Febiger ([1979], p. 92). Of particular significance is the association of certain E. coli strains with such serious diseases as hemolytic uremic syndrome (W. R. Gransden et al., "Further evidence associating hemolytic uremic syndrome with infection by verotoxin-producing Escherichia coli O157:H7," J. Infect. Dis., 154:522-534 [1986]), highlighting the importance of E. coli strains in severely debilitating UTI's.
In addition to E. coli, other members of the Enterobacteriaceae have been associated with UTI's. For example, Proteus is frequently isolated in UTI's in boys (Kunin, at pp. 47 and 92). Klebsiella pneumoniae is another important organism in urinary tract infections, as it has been reported to be the second most common pathogen isolated from UTI's (S. Falkow and J. Mekalanos, "The Enteric Bacilli and Vibrios," pp. 561-587 in B. D. Davis et al. (eds.), Microbiology, 4th ed., J. B. Lippincott Co., Philadelphia, 1990]). Indeed, of the Enterobacteriaceae, "80 to 95% of all isolates seen in a general hospital setting will be Escherichia coli, Klebsiella pneumoniae, or Proteus mirabilis." (J. J. Farmer et al., "Biochemical Identification of New Species and Biogroups of Enterobacteriaceae Isolated from Clinical Specimens," J. Clin. Microbiol., 21:46-76 [1985]). Undoubtedly, given the large number of specimens, a major proportion of these isolates are from UTI's. Other species of enteric bacteria are infrequently isolated from UTI's including such noted pathogens as Salmonella and Shigella.
Pseudomonas aeruginosa, an organism that is ubiquitous in the environment can infect almost any tissue or body site, including localized lesions in the urinary tract. UTI's due to P. aeruginosa are more common among the elderly (W. K. Joklik et al., Zinsser Microbiology, Appleton-Century-Crofts, Norwalk, Conn., 1984, p. 631-636). This organism is recognized as being particularly debilitating in patients with underlying disease or immunocompromised conditions.
In addition to the gram-negatives, various gram-positive organisms are commonly associated with UTI's. Enterococcus faecalis is a gram-positive coccus previously included within the genus Streptococcus. Like E. coli (and most species of Enterobacteriaceae), E. faecalis is a member of the normal gastrointestinal flora of humans and may also be found among the normal vaginal flora. Although E. faecalis is associated with various other diseases, UTI's are the most frequent diseases caused by this organism (R. C. Moellering, "The Enterococcus: A versatile pathogen," pp. 3-6, in Challenges in Gram-Positive Infection: A Global Perspective.TM., Healthmark [1988]). Treatment considerations are significant in E. faecalis disease, as this organism is resistant to a large number of antimicrobial agents. For example, E. faecalis is tolerant to a number of antimicrobials that are bactericidal against other bacteria. This high degree of antimicrobial resistance highlights the necessity of identifying this organism from UTI's.
Of the important gram-positive organisms, S. saprophyticus was relatively recently identified as a cause of UTI's (R. H. Latham et al., "Urinary tract infections in young adult women caused by Staphylococcus saprophyticus," J. Amer. Med. Assoc., 250:3063-3066 [1983]; and G. Wallmark et al., "Staphylococcus saprophyticus: A Frequent Cause of Urinary Tract Infection Among Female Outpatients," J. Infect. Dis., 138:791-797 [1978]). Prior to the association of this organism with UTI's, it was generally thought that coagulase-negative staphylococci were apathogenic when isolated from the urinary tract (see e.g., B. Hovelius and M.ang.rdh, "Staphylococcus Saprophyticus As a Common Cause of Urinary Tract Infections," Rev. Infect. Dis., 6:328-337, 1984). As S. saprophyticus is one of the most common organisms associated with UTI's in young women, the importance of this organism is now recognized. Importantly, not only are these organisms associated with UTI's, they have also been associated with serious infections such as pyelonephritis and sepsis (W. Lee et al., "Pyelonephritis and sepsis due to Staphylococcus saprophyticus," J. Infect. Dis., 155:1079-1080 [1987]). Unlike E. coli and the other enteric organisms, the reservoir for S. saprophyticus remains to be determined.
The organisms discussed above are most commonly associated with ascending infection (A. J. Schaeffer, "Cystitis and Pyelonephritis," pp. 418-435, in Youmans et al., (eds.), The Biologic and Clinical Basis of Infectious Disease, W. B. Saunders, [1986]). However, organisms may enter the urinary tract by direct extension from the gastrointestinal tract or through hematogenous spread. UTI's may also arise as infections secondary to bacteremia associated with extensive infection at other body sites (Schaeffer, at pp. 421-423). Hematogenous spread to the kidneys is more common with organisms such as Staphylococcus aureus, Candida sp., and Mycobacterium sp. Thus, organisms may gain access to the structures of the urinary tract through a variety of means, including surgical procedures and catheterization.
The following table lists the organisms commonly associated with hospital and community-acquired UTI's. Notably, a large proportion of these organisms are also residents of the normal gastrointestinal and/or urinary tracts and/or vagina. Table 3 lists the organisms more rarely isolated from UTI's.
TABLE 2 ______________________________________ Organisms Most Commonly Associated With UTI's Acquired In The Community and Hospital Settings Hospitalized Patients Outpatients Intensive Initial Recurrent Medical Care Units Organism Cases (%) Cases (%) Wards (%) (%) ______________________________________ E. coli .gtoreq.90 69 42 24 P. mirabilis 5 8 6 2 Klebsiella- 1 6 13 16 Enterobacter sp. Enterococcus 1 3 15 23 sp. Staphylo- 1 3 7 5 coccus sp. (coagulase negative) P. aeruginosa 0 &lt;1 6 17 S. marcesens 0 0 1 3 All other 2 11 10 10 organisms ______________________________________ *After Clarridge et al, p. 2.
TABLE 3 ______________________________________ Less Common And Unusual Agents Associated With Urinary Tract Infections* ______________________________________ Mycobacterium sp. Leptospira sp. H. influenzae G. vaginalis Acinetobacter sp. Alcaligenes sp. Pseudomonas sp. Citrobacter sp. N. gonorrhoea Salmonella sp. (including S. typhi) Shigella sp. .beta.-Hemolytic Streptococci Anaerobes C. trachomatis T. vaginalis S. haematobium Herpes Virus ______________________________________ *Koneman et al.,; Baron & Finegold; and Power & McCuen.
Although many organisms may be isolated from UTI's, the chances are good that the isolate will belong to one of the organisms listed in Table 2, highlighting the importance of identifying a relatively small number of organisms associated with UTI's.
An additional concern relates to the type of cultures isolated from the urinary tract. Pure cultures are most commonly associated with UTI's in the general population. However, mixed cultures are frequently observed in hospitalized patients. These mixed infections may present treatment problems, as the therapeutic regimen must be directed to all of the organisms involved. The frequency of mixed cultures is highlighted by a recent study cited by Orenga et al. (supra), in which De Montclos & Carret found that 25% of the urine cultures from hospitalized patients were mixed (De Montclos & Carret, "Optimisation de l'examen cytobacteriolique urinaire," Spectra Biologie, 92:49-53 (1992)). Importantly, mixed infections may also present diagnostic problems, as certain organisms may mask the presence of other species.
Due to the prevalence of UTI's, diagnosis of these infections is a common laboratory procedure. Various methods have been developed for the isolation, identification, and/or detection of the organisms most commonly associated with UTI's. Of these methods, there are two major categories: (1) culture methods, which utilize traditional microbiological culturing techniques to isolate, and then identify microorganisms based on their characteristic biochemical profiles, and for some species, their serological profiles; and (2) non-culture methods, which utilize various enzyme and other systems to detect the presence of infection.